Modifications and uses of conotoxin peptides

ABSTRACT

The present disclosure describes analog conotoxin peptides of the α-conotoxin peptide RgIA. These analog conotoxin peptides block the α9α10 subtype of the nicotinic acetylcholine receptor (nAChR) and can be used for treating pain and inflammation including inflammatory pain, cancer related pain, and neuropathic pain. The RgIA analogs described in the present invention include a variety of sequence modifications and chemical modifications that are introduced to improve the drug-like characteristics of RgIA analogs and thereby increase their therapeutic value.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/123,123 filed Nov. 7, 2014, the entire contents of which areincorporated by reference herein.

FIELD OF THE DISCLOSURE

The disclosure provides modified sequences of conotoxin peptides,pharmaceutical compositions of conotoxin peptides, and methods of usethereof for treating pain and other disorders.

BACKGROUND OF THE DISCLOSURE

Predatory marine snails in the genus Conus have venoms that are rich inneuropharmacologically active peptides (conotoxin peptides or cone snailproteins “CSP”). There are approximately 500 species in Conus, and amongthose that have been examined so far, a conserved feature is thepresence of α-conotoxin peptides in their venom. Native α-Conotoxinpeptides are highly disulfide cross-linked peptides with C1-C3 and C2-C4disulfide bonds.

Due to high sequence variability of their non-cysteine residues,α-conotoxins are extremely diverse and each Conus species has a uniquecomplement of α-conotoxin peptides. α-Conotoxin peptides are synthesizedas large precursors, and the mature toxin is generated by a proteolyticcleavage toward the C-terminus of the precursor. In contrast to thevariable inter-cysteine sequences of the mature toxins, the precursorsand the genes encoding them are quite conserved both among α-conotoxinpeptides in a given Conus species and from species to species.

α-Conotoxin peptides have generally been shown to be nicotinicacetylcholine receptor (nAChR) antagonists (McIntosh, et al., 1999;Janes, 2005; Dutton et al., 2001; Arias et al., 2000). nAChRs are agroup of acetylcholine gated ion channels that are part of the ligandgated ion channel superfamily. They are pentamers of transmembranesubunits surrounding a central ion conducting channel. Many differentsubunits have been identified, and most fall into two main subfamilies(α subunits and β subunits). The subunits can associate in variouscombinations in the receptor pentamers, leading to a diverse family ofreceptor subtypes. Most of the subtypes contain subunits from both the αand β subunit families, e.g., the human adult muscle subtype containstwo α subunits and a β subunit (in addition to a δ and an ε subunit),and the α4β2 central nervous system subtype is composed of α4 and β2subunits. Examples of nAChRs that are composed of only α subunits arethe α7 and α9 subtypes (homopentamers) and the α9α10 subtype (an all αheteropentamer). Phylogenetic analysis shows that the α7, α9, and α10subunits are more closely related to each other than they are to othernAChR subunits.

The α9 and α10 nAChR subunits are expressed in diverse tissues. In theinner ear α9α10 nAChRs mediate synaptic transmission between efferentolivocochlear fibers and cochlear hair cells. The α9 and α10 subunitsare also found in dorsal root ganglion neurons, lymphocytes, skinkeratinocytes, and the pars tuberalis of the pituitary. In addition, theα9 nAChR subunit is active in breast cancer. α-Conotoxin peptide RgIA(SEQ ID NO:1) has been shown to block α9α10 nAChR (Ellison, et al.,2006). Certain analogs of RgIA have also been shown to block α9α10 nAChRas demonstrated in US 2009/0203616, US 2012/0220539, and WO 2008/011006.

In general, the therapeutic potential of peptide drug candidates can beimproved either by formulation or by their non-covalent or covalentchemical modification. The practical utilization of peptides astherapeutics has been limited by relative low solubility andphysicochemical stability, both in formulation as drug products and invivo after administration to an animal or a human. Parenteral peptidedrugs, in particular, are rapidly cleared from circulation by kidneyfiltration or the reticuloendothelial system. They are also oftensusceptible to rapid degradation by circulating proteases. Finally,peptides can be immunogenic which can limit their therapeutic use due torisk of removal by antibodies or, in some instances, incidence ofinflammatory reactions (e.g., anaphylactic-like reactions). In addition,oral delivery of peptides is hampered by the lack of dedicated peptidetransporters in the intestines that allow the uptake of peptides oflengths greater than 2-4 amino acids, as well as the difficulty ofpassage though the low pH environment of the stomach.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to modifications to α-conotoxin peptidesincluding RgIA and RgIA analogs in order to increase their potential foruse in therapeutics for pain and inflammation. These changes includeamino acid modifications, which as used herein include deletions,substitutions, and additions. These changes also include attaching nonamino acid functional groups or molecules to the peptides such as fattyacid chains, acetyl groups, PEGylation, and/or glycosylation groups.Additional changes include RgIA analogs modified to containglycine-alanine N- to C-terminus bridges that effectively cyclize thepeptides. These approaches increase desirable drug-like propertiesincluding peptide stability in vitro and in vivo, increase theirhalf-life in circulation, increase their oral bioavailability such as byfacilitation of passage through the stomach and increase in absorption,and reduce renal/hepatic clearance once in circulation. Thesemodifications of analog conotoxin peptides are used to block the α9α10subtype of the nicotinic acetylcholine receptor (nAChR) with very highselectivity and affinity and thereby produce analgesic andanti-inflammatory effects in inflammatory, neuropathic, cancer, andother disease states.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1G show modifications to RgIA to constrain the isomerization ofthe conserved aspartate residue in the conserved sequence Asp-Pro-Arg.Isomerization is shown in FIG. 1A. Modifications are shown in FIGS.1B-1G.

FIG. 2A and FIG. 2B show increased stability of CSP-2 (SEQ ID NO:3) andCSP-4 (SEQ ID NO:5), respectively, in serum following amidation of theC-terminus. FIG. 2A shows CSP-2 and CSP-2-NH2 stability in rat serum attime 0-2 hours; CSP-2-NH2 shows increased stability compared to CSP-2over time. FIG. 2B shows CSP-4 and CSP-4-NH2 stability in human plasma(Citrate) during time 0-24 hours; CSP-4-NH2 shows increased stabilitycompared to CSP-4 over time. Measurements taken at time=0 were takenwithin 30 seconds of mixing peptide with serum.

FIGS. 3A-3D show connectivity schemes of bridges in RgIA analogs. X andY represent substitution of the amino acid residue with a naturally orunnaturally occurring amino acid in the R- or S-configuration (i.e., D-or L-amino acids) that are then coupled for bridge formation. In SEQ IDNO:25, the original amino acid at X and Y is cysteine, and the bridge isa disulfide bridge. The coupler can be a peptide, C1-C5 alkane,dialkylether, dialkyl thioether, repeating ethoxyether, alkene; or thebridging moiety may be connected directly to the peptide backbone. Thebridging moiety can be diselenide, disulfide, 5- or 6-memberedheterocyclic rings, an alkene, amide bond, carbamate, urea, thiourea,sulfonamide, sulfonylurea. Examples of 5-membered rings include1,2-diazoles, oxazoles, and thiazoles, 1,3-diazoles, oxazoles andthiazoles, 1,2,3-triazoles, 1,2,4-triazoles, tetrazoles. Connectivity tothe coupler units can be through the adjacent ring atoms (i.e. 1,2-, or1,5-) or separated by one ring atom (i.e. 1,3- or 1,4-).

FIG. 4 shows four examples of peptides each bridged with one cysteinedisulfide and one lactam bridge, the latter based on glutamic acid andlysine. Standard peptide synthesis methods are employed for the mainpeptide chain; the bridging amide can be formed selectively via use ofprotecting groups orthogonal to the chemistry employed for the mainpeptide chain. X designates replacement of the cysteine residue with anaturally or unnaturally occurring amino acid. In the four examplesshown here, cysteine is replaced with glutamic acid at position 2 andwith lysine at position 8 (lactam #1), cysteine is replaced with lysineat position 2 and with glutamic acid at position 8 (lactam #2), cysteineis replaced with glutamic acid at position 3 and with lysine at position12 (lactam #3), cysteine is replaced with lysine at position 3 and withglutamic acid at position 12 (lactam #4).

FIG. 5 shows two examples of peptides bridged with one cysteinedisulfide and one triazole bridge, the latter formed from 1, 3-dipolarcycloaddition reaction (i.e. “Click chemistry”) from modified aminoacids. X designates a modified amino acid that replaces a cysteineresidue for bridge formation; in the examples given here, the modifiedamino acids are (S)-propargyl glycine and (S)-azidonorvaline, which havebeen coupled to form the 1,2,3-triazole ring shown.

FIG. 6 shows pharmacokinetics of CSP-4-NH2 and lipidated analogs ofCSP-4-NH2 measured in plasma. CSP-4-NH2 (Δ), C12-CSP-4-NH2 (◯) andC16-CSP-4-NH2 (▪) were administered subcutaneously to mice (500 mg/Kg).Mice, n=3. Error bars represent Standard Error of the Mean (S.E.M.) ofthe three samples. CX- represents a lipid moiety of length X conjugatedto the CSP.

FIGS. 7A AND 7B show efficacy of lipidated CSP-4-NH2 analogs incapsaicin model of neuropathic pain. Subcutaneous administration of 500mg/kg C12-CSP-4-NH2 (FIG. 7A) and C16-CSP-4-NH2 (FIG. 7B) was effectivein reducing capsaicin-induced thermal hyperalgesia (Hargreaves test). *designates values significantly different than vehicle. P<0.05. Rats,n=6. Error bars represent S.E.M. of the six samples.

FIG. 8 shows efficacy of lipidated analog, C12-CSP-4-NH2 in achemotherapy-induced neuropathy model in rats. Mechanical hyperalgesia(t=0; Randal Selitto test) was reduced following a single subcutaneousinjection of C12-CSP-4-NH2 (500 mg/Kg) that lasted 53 hours. *designates values significantly different than at time 0. P<0.05. Rats,n=8. Error bars represent S.E.M. of the eight samples.

FIG. 9 shows extended pharmacotherapeutic effect of PEGylated analog ofCSP-4-NH2 in chemotherapy-induced neuropathic pain model.PEG-SVA-CSP-4-NH2, was effective in reducing chemotherapy-inducedneuropathic pain (CINP)-mechanical hyperalgesia (t=0; Randal-Selittotest) following a single subcutaneous injection of PEG-SVA-CSP-4-NH2(500 mg/Kg). Effect lasted 75 hours. * designates values significantlydifferent than at time 0. P<0.05. Rats, n=8. Error bars represent S.E.M.of the eight samples.

DETAILED DESCRIPTION

The present disclosure relates to the α-conotoxin peptide RgIA (SEQ IDNO:1), conotoxin peptides that are analogs of the α-conotoxin peptideRgIA, as well as modifications thereof (collectively “RgIA analogs”herein). These RgIA analogs block the α9α10 subtype of the nicotinicacetylcholine receptor (nAChR) and can be used to treat pain andinflammation. These pain conditions include musculoskeletal pain,inflammatory pain, cancer pain, neuropathic pain syndromes includingdiabetes neuropathic pain, chemotherapy-induced pain, postherpeticneuralgia, idiopathic neuropathic peripheral pain, phantom limb pain,orthopedic pain including osteoarthritis, andautoimmune/inflammatory-induced pain including rheumatoid arthritispain. The RgIA analogs can also be used in further drug development asdescribed herein.

Marine snails produce a number of peptides that have neurotoxic effectson prey. Peptides from the genus Conus typically range from 12 to 30amino acids in length and contain 4 or more cysteine residues; theconotoxins of the subtype alpha contain and form two disulfide bonds ina C1-C3 and C2-C4 connectivity. α-Conotoxin peptides bind nAChRs. One ofthese, RgIA (SEQ ID NO:1), is selective for α9α10 nAChRs that have beendemonstrated to have analgesic properties in several models ofneuropathic pain and inflammation. In addition to the conserved cysteineresidues, the proline residue is also conserved and the DPR regionfunctions in binding to the α9α10 nAChR. The arginine residue atposition 9 is associated with increased selectivity for the human α9α10nAChR.

Previously described (PCT/US2014/040374) analogs of RgIA that havedesired drug-like characteristics such as increased affinity for thehuman α9α10 nAChR target compared to the parent RgIA and increased invitro and in vivo stability (Table 1). Previously described RgIA analogsare also disclosed in U.S. Pat. Nos. 6,797,808; 7,279,549; 7,666,840;7,902,153; 8,110,549; 8,487,075; and 8,735,541; and in U.S. patentapplication Ser. Nos. 12/307,953 and 13/289,494; the sequences of whichare incorporated by reference. The present disclosure also relates toadditional analogs of RgIA as listed in Table 2.

The present disclosure describes a series of modifications that can bemade to RgIA analogs, including those listed in Tables 1 and 2, toimprove their drug like characteristics for their therapeutic useincluding as analgesics.

TABLE 1 Analog Sequence SEQ ID NO. CSP-P GCCSDPRCRYRCR 1 CSP-1GCCSDPRCRX12RCR 2 CSP-2 GCCTDPRCX11X12QCR 3 CSP-3 GCCTDPRCX11X12QCRRR 4CSP-4 GCCTDPRCX11X12QCY 5 CSP-5 GX13CTDPRX13X11X12QCR 6 CSP-6GCCTDPRCRX12QCF 7 CSP-7 GCCTDPRCRX12QCY 8 CSP-8 GCCTDPRCRX12QCW 9 X11= Citrulline X12 = 3-iodo-Tyrosine X13 = Selenocysteine

TABLE 2 Sequence SEQ ID NO. GCCTDPRCX21X12QCYRR 222 GCCTDPRCX21X12QCRRY223 GCCTDPRCX21X12QCF 224 GCCTDPRCX21X12QCW 225 GCCTDPRCX21X12QCYY 226GCCTDPRCX21X12QCYR 227 GCCTDPRCRX12QCRRR 228 GCCTDPRCRX12QCYRR 229GCCTDPRCRX12QCRRY 230 GCCTDPRCRX12QCYY 231 GCCTDPRCRX12QCYR 232GCCSDPRCNYDHPEIC 233 GCCSDPRCNYDHPEIC-amide 234 GCCSHPACSVNHPELC 235GCCSHPACSVNHPELC-amide 236 GCCTDPRCRYRCR 237 GCCSDX14QRCRYRCR 238GCCTDX14RCRYRCR 239 GCCSDPRCRX15RCR 240 GCCTDPRCRX15RCR 241X15GCCSDX14RCRX15RCR 242 X15GCCTDX14RCRX15RCR 243 GCCSDPRCX16YRCR 244GCCSDPRCX21X12RCR 245 GX13CTDPRX13X21X12QCK 246 GX13CSDPRX13RYRCR 247GCCTDPRCX21X12RCR 248 GCCSDPRCX21YRCR 249 GCCSDPRCRYQCR 250GCCSDPRCFWRCR 251 GX17CSDPRCRYRCR 252 GCCADPRCRYRCR 253 GCCYDPRCRYRCR254 GCCSDPRX17RYRCR 255 GCCSDPRCGYRCR 256 GCCSDPRCAYRCR 257GCCSDPRCVYRCR 258 GCCSDPRCLYRCR 259 GCCSDPRCIYRCR 260 GCCSDPRCMYRCR 261GCCSDPRCFYRCR 262 GCCSDPRCWYRCR 263 GCCSDPRCPYRCR 264 GCCSDPRCSYRCR 265GCCSDPRCTYRCR 266 GCCSDPRCCYRCR 267 GCCSDPRCYYRCR 268 GCCSDPRCNYRCR 269GCCSDPRCQYRCR 270 GCCSDPRCDYRCR 271 GCCSDPRCEYRCR 272 GCCSDPRCKYRCR 273GCCSDPRCHYRCR 274 GCCSDPRCRFRCR 275 GCCSDPRCRYHCR 276 GCCSDPRCX18X12RCR277 GCCSDPRCRYRC 278 GCCSEPRCRYRCR 279 GCCSDVRCRYRCR 280 GCCSDPRCAYRCR281 GCCSHPACRYRCR 282 GCCSDPRCX19YRCR 283 ACCSDRRCRWRC 284FDGRNAPADDKASDLIAQIVRRACCSDRRCRWRCG 285 X15GCCSX14RCRX15RCR 286SNKRKNAAMLDMIAQHAIRGCCSDPRCRYRCR 287 DECCSNPACRVNNPHV 288SDGRNVAAKAFHRIGRTIRDECCSNPACRVNNPHVCRRR 289 DECCSNPACRLNNPHACRRR 290DX20CCSNPACRLNNPHACRRR 291 DECCSNX14ACRLNNPHACRRR 292X20DX20CCSNX14ACRLNNPHACRRR 293 DECCSNPACRLNNX14HACRRR 294X20DX20CCSNPACRLNX14HACRRR 295 DECCSNX14ACRLNNX14HACRRR 296X20DX20CCSNX14ACRLNNX14HACRRR 297 DECCSNPACRLNNPHVCRRR 298DX20CCSNPACRLNNPHVCRRR 299 DECCSNX20ACRLNNPHVCRRR 300X20DX20CCSNX14ACRLNPHVCRRR 301 DECCSNPACRLNNX14HVCRRR 302X20DX20CCSNX14ACRLNNPHVCRRR 303 DECCSNX14ACRLNNX14HVCRRR 304X20DX20CCSNX14ACRLNNX14HVCRRR 305 GCCSHPACNVDHPEIC 306MFTVFLLVVLATTVVSFTSDRAFRGRNSAANDK 307 RSDLAALSVRRGCCSHPACSVNHPELCGRRRECCTNPVCHAEHQHELCARRR 308 ECCTNPVCHAX21HQELCARRR 309ECCTNPVCHAX21HQX21LCARRR 310 ECCTNPVCHAX12HQX21LCARRR 311X21CCTNPVCHAEHQHELCARRR 312 X21CCTNPVCHAX21HQELCARRR 313X21CCTNPVCHAX21HQX21LCARRR 314 X21CCTNPVCHAX12HQX21LCARRR 315GCCSHPVCSAMSPIC 316 GCCSHPVCSAMSX1IC 317 GCCSHX14VCSAMSX1IC 318GCCSHX14VCSAMSPIC 319 X1 = des-X1, Arg or citrulline X11 = CitrullineX12 = 3-iodo-Tyrosine X13 = Selenocysteine X14 = hydroxy-Pro X15= mono-halo Tyr including iodo-Tyr, bromo-Tyr X16 = homo-Arg orornithine X17 = homocysteine X18 = omega-nitro-Arg X19 = D-Arg X20= γ-carboxy-Glu (Gla) X21 = 7-carboxy-Glu

In various embodiments, analog RgIA analogs disclosed herein have theformula X10 X6 X7 X3 D P R X8 X1 X12 X4 X9 X5 (SEQ ID NO:10), wherein X1is des-X1, Arg or citrulline; X3 is des-X3, Ser, or Thr; X4 is des-X4,Arg or Gln; X5 is des-X5, Arg, Tyr, Phe, Trp, Tyr-Tyr, Tyr-Arg,Arg-Arg-Arg, Arg-Arg, Arg-Tyr, Arg-Arg-Tyr, or Tyr-Arg-Arg; X6 isdes-X6, Cys, or selenocysteine; X7 is des-X7, Cys, or selenocysteine; X8is des-X8, Cys, or selenocysteine; X9 is des-X9, Cys, or selenocysteine;and X10 is des-X10 or Gly. In one embodiment, X10 is Gly, X6 is Cys orselenocysteine, X7 is Cys, X3 is Ser or Thr; X8 is Cys orselenocysteine, X1 is Arg or citrulline, X4 is Arg or Gin, X9 is Cys,and X5 is Arg, Tyr, Phe, Trp, or Arg-Arg-Arg (SEQ ID NO:11). In oneembodiment, X10 is Gly, X6 is Cys or selenocysteine, X7 is Cys, X3 isThr, X8 is Cys or selenocysteine, X1 is Arg or citrulline, X4 is Gin, X9is Cys, and X5 is Arg or Tyr (SEQ ID NO:12).

In various embodiments modifications to the RgIA and its analogs aremade so as to prevent the isomerization of the conserved aspartateresidue to isoaspartate in the conserved tripeptide sequence“Asp-Pro-Arg”. This isomerization is shown in FIG. 1A. This approachprevents this isomerization and results in stable RgIA analogs thatmaintain their pharmacological properties of high affinity and highselectivity in binding to the intended target, namely α9α10 nAChRs.Therefore, despite the small globular size of RgIA conotoxin peptides,the peptide bond replacements and the proposed strategies presentedhereby result in bioactive, potent, and more stable peptides. Threedifferent chemical approaches are used and evaluated. In the firstteaching, the aspartic acid is replaced with amino malonic acid (FIG.1B; 2-amino propandioic acid), which is equivalent to an aspartic acidwith a shortened side chain. This derivative with the shortened sidechain cannot form 5-membered succinic acid anhydride intermediate thatis necessary for production of the isomer. Synthesis can be accomplishedvia standard peptide chemistry using a suitably protected amino malonicacid (e.g., FIG. 1C).

In the next two teachings, a non-peptide bond is engineered to join theaspartic acid replacement and the proline via N-alkylation of theproline; both examples are non-hydrolysable and therefore notsusceptible to isomerization.

The second approach replaces the peptide-chain carbonyl group ofaspartic acid with a methylene group (FIG. 1D) to afford a ‘reducedpeptide bond’. This can be prepared by alkylating the proline with anappropriately protected Asp replacement such as(3S)-4-bromo-3-[[(1,1-dimethylethoxy)carbonyl]amino]-butanoic acid (FIG.1E) which itself is incorporated into the peptide chain via standardpeptide chemistry.

The third approach replaces the peptide chain carbonyl group of asparticacid with a ketomethyl group (FIG. 1F) which is the equivalent ofinserting a methylene group in the peptide chain between Asp and Pro.This can be prepared by alkylating the proline with an appropriatelyprotected Asp replacement such as1,1-dimethylethyl-(3S)-5-chloro-4-oxo-3-[[(phenylmethoxy)carbonyl]amino]-pentanoate(FIG. 1G), which itself is incorporated into the peptide chain viastandard peptide chemistry.

In various embodiments, amino acid modifications can increase peptidestability by replacement of amino acid residues that may be prone toenzymatic cleavage. Such modifications include: replacement of anyL-amino acid with the corresponding D-amino acid; replacement of Glywith a neutral amino acid, including Val, Nor-Val, Leu, or Ile;replacement of Arg with His or Lys; replacement of Pro with Gly;replacement of Gly with Pro, and/or replacement of cysteine withselenocysteine. Illustrative peptide sequences with such modificationsare described in Table 3.

TABLE 3 Modified peptide sequences with amino acid modificationsSequence SEQ ID NO. GCCSDPRCRYRCH 30 GCCSDPRCRYRCK 31 GCCSDPRCRX22RCR 32X23CCSDPRCRYRCR 33 X24CCSDPRCRYRCR 34 GCCSDX25RCRYRCR 35 GCCSX26PRCRYRCR36 GCCSDPX27CRYRCR 37 GCCSX26X25RCRYRCR 38 GCCSDX25X27CRYRCR 39GCCSX26X25X27CRYRCR 40 GCCSDPRCRYHCR 41 GCCSDPRCRYKCR 42 PCCSDPRCRYRCR43 GCCSDPRCRX12RCH 44 GCCSDPRCRX12RCK 45 GCCSDPRCRX22RCH 46X23CCSDPRCRX12RCR 47 X24CCSDPRCRX12RCR 48 GCCSDX25RCRX12RCR 49GCCSX26PRCRX12RCR 50 GCCSDPX27CRX12RCR 51 GCCSX26X25RCRX12RCR 52GCCSDX25X27CRX12RCR 53 GCCSX26X25X27CRX12RCR 54 GCCSDPRCRX12HCR 55GCCSDPRCRX12KCR 56 PCCSDPRCRX12RCR 57 GCCSDPRCHX12RCR 58 GCCSDPRCKX12RCR59 GCCTDPRCRX12RCH 60 GCCTDPRCRX12RCK 61 GCCTDPRCRX22RCR 62X23CCTDPRCRX12RCR 63 X24CCTDPRCRX12RCR 64 GCCTDX25RCRX12RCR 65GCCTX26PRCRX12RCR 66 GCCTDPX27CRX12RCR 67 GCCTX26X25RCRX12RCR 68GCCTDX25X27CRX12RCR 69 GCCTX26X25X27CRX12RCR 70 GCCTDPRCRX12HCR 71GCCTDPRCRX12KCR 72 PCCTDPRCRX12RCR 73 GCCTDPRCHX12RCR 74 GCCTDPRCKX12RCR75 GCCTDPRCX11X12QCHRR 76 GCCTDPRCX11X12QCKRR 77 GCCTDPRCX11X12QCRHR 78GCCTDPRCX11X12QCRKR 79 GCCTDPRCX11X12QCRRH 80 GCCTDPRCX11X12QCRRK 81GCCTDPRCX11X22QCRRR 82 X23CCTDPRCX11X12QCRRR 83 X24CCTDPRCX11X12QCRRR 84GCCTDX25RCX11X12QCRRR 85 GCCTX26PRCX11X12QCRRR 86 GCCTDPX27CX11X12QCRRR87 GCCTX26X25RCX11X12QCRRR 88 GCCTDX25X27CX11X12QCRRR 89GCCTX26X25X27CX11X12QCRRR 90 PCCTDPRCX11X12QCRRR 91 GCCTDPRCX11X22QCY 92GCCTDPRCX11X12QCX22 93 X23CCTDPRCX11X12QCY 94 X24CCTDPRCX11X12QCY 95GCCTDX25RCX11X12QCY 96 GCCTX26PRCX11X12QCY 97 GCCTDPX27CX11X12QCY 98GCCTX26X25RCX11X12QCY 99 GCCTDX25X27CX11X12QCY 100GCCTX26X25X27CX11X12QCY 101 PCCTDPRCX11X12QCY 102 GX13CTDPRX13X11X12QCH103 GX13CTDPRX13X11X12QCK 104 GX13CTDPRX13X11X22QCR 105X23X13CTDPRX13X11X12QCR 106 X24X13CTDPRX13X11X12QCR 107GX13CTDX25RX13X11X12QCR 108 GX13CTX26PRX13X11X12QCR 109GX13CTDPX27X13X11X12QCR 110 GX13CTX26X25RX13X11X12QCR 111GX13CTDX25X27X13X11X12QCR 112 GX13CTX26X25X27X13X11X12QCR 113PX13CTDPRX13X11X12QCR 114 GCCTDPRCRX22QCF 115 X23CCTDPRCRX12QCF 116X24CCTDPRCRX12QCF 117 GCCTDX25RCRX12QCF 118 GCCTX26PRCRX12QCF 119GCCTDPX27CRX12QCF 120 GCCTX26X25RCRX12QCF 121 GCCTDX25X27CRX12QCF 122GCCTX26X25X27CRX12QCF 123 PCCTDPRCRX12QCF 124 GCCTDPRCRX22QCY 125GCCTDPRCRX12QCX22 126 X23CCTDPRCRX12QCY 127 X24CCTDPRCRX12QCY 128GCCTDX25RCRX12QCY 129 GCCTX26PRCRX12QCY 130 GCCTDPX27CRX12QCY 131GCCTX26X25RCRX12QCY 132 GCCTDX25X27CRX12QCY 133 GCCTX26X25X27CRX12QCY134 PCCTDPRCRX12QCY 135 GCCTDPRCRX22QCW 136 X23CCTDPRCRX12QCW 137X24CCTDPRCRX12QCW 138 GCCTDX25RCRX12QCW 139 GCCTX26PRCRX12QCW 140GCCTDPX27CRX12QCW 141 GCCTX26X25RCRX12QCW 142 GCCTDX25X27CRX12QCW 143GCCTX26X25X27CRX12QCW 144 PCCTDPRCRX12QCW 145 GX13CTDPRCX11X12QCY 146GCX13TDPRCX11X12QCY 147 GCCTDPRX13X11X12QCY 148 GCCTDPRCX11X12QX13Y 149GX13CTDPRX13X11X12QCY 150 GCX13TDPRCX11X12QX13Y 151GX13X13TDPRCX11X12QCY 152 GCCTDPRX13X11X12QX13Y 153GX13CTDPRCX11X12QX13Y 154 GX13X13TDPRX13X11X12QCY 155GCX13TDPRX13X11X12QX13Y 156 GX13CTDPRX13X11X12QX13Y 157GX13X13TDPRCX11X12QX13Y 158 GX13X13TDPRX13X11X12QX13Y 159GX13CTDPRCRX12QCY 160 GCX13TDPRCRX12QCY 161 GCCTDPRX13RX12QCY 162GCCTDPRCRX12QX13Y 163 GX13CTDPRX13RX12QCY 164 GCX13TDPRCRX12QX13Y 165GX13X13TDPRCRX12QCY 166 GCCTDPRX13RX12QX13Y 167 GX13CTDPRCRX12QX13Y 168GX13X13TDPRX13RX12QCY 169 GCX13TDPRX13RX12QX13Y 170GX13CTDPRX13RX12QX13Y 171 GX13X13TDPRCRX12QX13Y 172GX13X13TDPRX13RX12QX13Y 173 X11 = Citrulline X12 = 3-iodo-Tyrosine X13= Selenocysteine X14 = hydroxy-Pro X15 = mono-halo Tyr includingiodo-Tyr, bromo-Tyr X16 = homo-Arg or ornithine X17 = homocysteine X18= omega-nitro-Arg X19 = D-Arg X20 = γ-carboxy-Glu (Gla) X21= 7-carboxy-Glu X22 = O-phospho-Tyr, O-sulfo-Tyr, or O-fluoro-Tyr X23= mono-fluoro-Glycine X24 = di-fluoro-Glycine X25 = D-Pro X26 = D-AspX27 = D-Arg

In various embodiments, linkers are added to RgIA analog peptides usingstandard peptide chemistry. The addition of one or more linkers aroundconserved regions that have been shown to be involved in targetrecognition increases the stability and binding affinity of RgIAanalogs. Illustrative peptide sequences with such changes are describedin Table 4.

TABLE 4 Peptide sequences with added of linkers Sequence SEQ ID NO.X10X6X7X3[AEA]DPRX8X1X2X4X9X5 174 X10X6X7X3D[AEA]PRX8X1X2X4X9X5 175X10X6X7X3DP[AEA]RX8X1X2X4X9X5 176 X10X6X7X3DPR[AEA]X8X1X2X4X9X5 177X10X6X7X3[AEEA]DPRX8X1X2X4X9X5 178 X10X6X7X3D[AEEA]PRX8X1X2X4X9X5 179X10X6X7X3DP[AEEA]RX8X1X2X4X9X5 180 X10X6X7X3DPR[AEEA]X8X1X2X4X9X5 181X10X6X7X3[AEEEA]DPRX8X1X2X4X9X5 182 X10X6X7X3D[AEEEA]PRX8X1X2X4X9X5 183X10X6X7X3DP[AEEEA]RX8X1X2X4X9X5 184 X10X6X7X3DPR[AEEEA]X8X1X2X4X9X5 185X1 = des-X1, Arg, citrulline, or ω-nitro-Arg X2 = des-X2, Tyr, ormono-iodo-Tyr X3 = des-X3, Ser, or Thr X4 = des-X4, Arg or Gln X5= des-X5, Arg, Tyr, phenylalanine (Phe or F), tryptophan (Trp or W),Tyr-Tyr, Tyr-Arg, Arg-Arg-Arg, Arg-Arg, Arg-Tyr, Arg-Arg-Tyr, orTyr-Arg-Arg X6 = des-X6, Cys, or selenocysteine X7 = des-X7, Cys, orselenocysteine X8 = des-X8, Cys, or selenocysteine X9 = des-X9, Cys, orselenocysteine X10 = des-X10 or Gly AEA = 2-amino ethoxyacetic acid AEEA= 2-[2-[ethoxy]ethoxy]acetic acid AEEEA= 2-[2-[2-[ethoxy]ethoxy]ethoxy]acetic acid

In various embodiments the RgIA analogs may have a modification to theN-terminus and/or the C-terminus. Such modifications include: acylationof the N-terminal Gly and/or amidation of the C-terminus (Table 5);acylation of the N-terminal Gly, replacement of the C-terminal aminoacid with the corresponding D-isomer (indicated by a lower case letter),and/or amidation of the C-terminus (Table 6). Selected illustrativepeptide sequences with these changes are shown in Tables 5 and 6.

TABLE 5 Peptide sequences with modification of the N-terminus ormodification of the N- and C-terminus Sequence SEQ ID NO.Ac-GCCSDPRCRYRCR 186 Ac-GCCSDPRCRX3RCR 187 Ac-GCCTDPRCX2X3QCR 188Ac-GCCTDPRCX2X3QCRRR 189 Ac-GCCTDPRCX2X3QCY 190 Ac-GX4CTDPRX4X2X3QCR 191Ac-GCCTDPRCRX3QCF 192 Ac-GCCTDPRCRX3QCY 193 Ac-GCCTDPRCRX3QCW 194Ac-GCCSDPRCRYRCR-amide 195 Ac-GCCSDPRCRX3RCR-amide 196Ac-GCCTDPRCX2X3QCR-amide 197 Ac-GCCTDPRCX2X3QCRRR-amide 198Ac-GCCTDPRCX2X3QCY-amide 199 Ac-GX4CTDPRX4X2X3QCR-amide 200Ac-GCCTDPRCRX3QCF-amide 201 Ac-GCCTDPRCRX3QCY-amide 202Ac-GCCTDPRCRX3QCW-amide 203

TABLE 6 Peptide sequences with replacement of the C-terminal L-aminoacid with a D-amino acid and modification of the N-terminus ormodification of the N- and C-terminus Sequence SEQ ID NO.Ac-GCCSDPRCRYRCr 204 Ac-GCCSDPRCRX3RCr 205 Ac-GCCTDPRCX2X3QCr 206Ac-GCCTDPRCX2X3QCRRr 207 Ac-GCCTDPRCX2X3QCy 208 Ac-GX4CTDPRX4X2X3QCr 209Ac-GCCTDPRCRX3QCf 210 Ac-GCCTDPRCRX3QCy 211 Ac-GCCTDPRCRX3QCw 212Ac-GCCSDPRCRYRCr-amide 213 Ac-GCCSDPRCRX3RCr-amide 214Ac-GCCTDPRCX2X3QCr-amide 215 Ac-GCCTDPRCX2X3QCRRr-amide 216Ac-GCCTDPRCX2X3QCr-amide 217 Ac-GX4CTDPRX4X2X3QCr-amide 218Ac-GCCTDPRCRX3QCf-amide 219 Ac-GCCTDPRCRX3QCy-amide 220Ac-GCCTDPRCRX3QCw-amide 221

In various embodiments RgIA analogs may be modified by addition ofbridges such as lactam bridges or triazole bridges. As an example, FIGS.3-5 show bridge structures formed by modifications to the peptide of SEQID NO:25. FIGS. 3A-3C show three different connectivity schemes forbridges in RgIA analogs. In the connectivity schemes for bridges asapplied to SEQ ID NO:25, X designates cysteine residues that are eachsubstituted with a naturally or unnaturally occurring amino acidresidue. Bridges #1 and #2 are formed from bridging moieties as shown inFIG. 3D. For a given peptide, bridge #1 and #2 may be formed from thesame or different bridging moieties. RgIA analogs may have both bridges#1 and #2 formed from disulfide bridges.

The RgIA analogs may have either one or both of the disulfide bridgesreplaced by a lactam bridge. FIG. 4 shows examples of 4 configurationsof such lactam bridge replacements in RgIA analog CSP-4-NH2 (SEQ ID NO:25). The X at positions 2, 3, 8, and 12 designates a cysteine residuereplaced with a different natural amino acid or with an unnatural aminoacid. Standard peptide synthesis methods are employed for the mainpeptide chain; the bridging amide can be formed selectively via use ofprotecting groups orthogonal to the chemistry employed for the mainpeptide chain.

The RgIA analogs may also have one cysteine disulfide and one triazolebridge. Each of the cysteine residues are replaced with an amino acidthat is a bridge precursor component and contains an alkyne group or anazide group in its side chain, wherein the alkyne group and azide groupare coupled to form a 1,2,3-triazole via 1,3-dipolar cycloadditionchemistry. The triazole bridge is formed from a 1,3 dipolarcycloaddition reaction, e.g., “click chemistry.” FIG. 5 shows examplesof 4 configurations of such triazole bridge replacements in RgIA analogCSP-4-NH2 (SEQ ID NO:25). In the examples given here, each X in thepeptide represents a cysteine residue replaced with (S)-propargylglycine or (S)-azidonorvaline.

“Variants” of RgIA analogs disclosed herein include peptides having oneor more amino acid additions, deletions, stop positions, orsubstitutions, as compared to an analog conotoxin peptide disclosedherein.

An amino acid substitution can be a conservative or a non-conservativesubstitution. Variants of RgIA analogs disclosed herein can includethose having one or more conservative amino acid substitutions. As usedherein, a “conservative substitution” involves a substitution found inone of the following conservative substitutions groups: Group 1: alanine(Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T);Group 2: aspartic acid (Asp or D), glutamic acid (Glu or E); Group 3:asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg orR), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile orI), leucine (Leu or L), methionine (Met or M), valine (Val or V); andGroup 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trpor W).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, sulfur-containing). For example, analiphatic grouping may include, for purposes of substitution, Gly, Ala,Val, Leu, and Ile. Other groups containing amino acids that areconsidered conservative substitutions for one another include:sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, and Gln; smallaliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, andGly; polar, negatively charged residues and their amides: Asp, Asn, Glu,and Gln; polar, positively charged residues: His, Arg, and Lys; largealiphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and largearomatic residues: Phe, Tyr, and Trp. Additional information is found inCreighton (1984) Proteins, W.H. Freeman and Company.

Variants of analog conotoxin peptide sequences disclosed or referencedherein also include sequences with at least 70% sequence identity, atleast 80% sequence identity, at least 85% sequence, at least 90%sequence identity, at least 95% sequence identity, at least 96% sequenceidentity, at least 97% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity to a peptide sequencedisclosed or referenced herein. More particularly, variants of the RgIAanalogs disclosed herein include peptides that share: 70% sequenceidentity with any of SEQ ID NO:1-319; 80% sequence identity with any ofSEQ ID NO:1-319; 81% sequence identity with any of SEQ ID NO:1-319; 82%sequence identity with any of SEQ ID NO:1-319; 83% sequence identitywith any of SEQ ID NO:1-319; 84% sequence identity with any of SEQ IDNO:1-319; 85% sequence identity with any of SEQ ID NO:1-319; 86%sequence identity with any of SEQ ID NO:1-319; 87% sequence identitywith any of SEQ ID NO:1-319; 88% sequence identity with any of SEQ IDNO:1-319; 89% sequence identity with any of SEQ ID NO:1-319; 90%sequence identity with any of SEQ ID NO:1-319; 91% sequence identitywith any of SEQ ID NO:1-319; 92% sequence identity with any of SEQ IDNO:1-319; 93% sequence identity with any of SEQ ID NO:1-319; 94%sequence identity with any of SEQ ID NO:1-319; 95% sequence identitywith any of SEQ ID NO:1-319; 96% sequence identity with any of SEQ IDNO:1-319; 97% sequence identity with any of SEQ ID NO:1-319; 98%sequence identity with any of SEQ ID NO:1-319; or 99% sequence identitywith any of SEQ ID NO:1-319.

“% sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness between peptidesequences as determined by the match between strings of such sequences.“Identity” (often referred to as “similarity”) can be readily calculatedby known methods, including those described in: Computational MolecularBiology (Lesk, A. M., ed.) Oxford University Press, NY (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994);Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) AcademicPress (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,J., eds.) Oxford University Press, NY (1992). Preferred methods todetermine sequence identity are designed to give the best match betweenthe sequences tested. Methods to determine sequence identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of thesequences can also be performed using the Clustal method of alignment(Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also includethe GCG suite of programs (Wisconsin Package Version 9.0, GeneticsComputer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul,et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc.,Madison, Wis.); and the FASTA program incorporating the Smith-Watermanalgorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.](1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher:Plenum, New York, N.Y. Within the context of this disclosure it will beunderstood that where sequence analysis software is used for analysis,the results of the analysis are based on the “default values” of theprogram referenced. As used herein “default values” will mean any set ofvalues or parameters which originally load with the software when firstinitialized.

“D-substituted analogs” include RgIA analogs disclosed herein having oneor more L-amino acids substituted with D-amino acids. The D-amino acidcan be the same amino acid type as that found in the analog sequence orcan be a different amino acid. Accordingly, D-analogs are also variants.

“Modifications” include RgIA analogs disclosed herein wherein one ormore amino acids have been replaced with a non-amino acid component, orwhere the amino acid has been conjugated to a functional group or afunctional group has been otherwise associated with an amino acid. Themodified amino acid may be, e.g., a glycosylated amino acid, a PEGylatedamino acid (covalent and non-covalent attachment or amalgamation ofpolyethylene glycol (PEG) polymers), a farnesylated amino acid, anacetylated amino acid, an acylated amino acid, a biotinylated aminoacid, a phosphorylated amino acid, an amino acid conjugated to a lipidmoiety such as a fatty acid, or an amino acid conjugated to an organicderivatizing agent. The presence of modified amino acids may beadvantageous in, for example, (a) increasing polypeptide serum half-lifeand/or functional in vivo half-life, (b) reducing polypeptideantigenicity, (c) increasing polypeptide storage stability, (d)increasing peptide solubility, (e) prolonging circulating time, and/or(f) increasing bioavailability, e.g. increasing the area under the curve(AUC_(sc)). Amino acid(s) can be modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means. The modified amino acidcan be within the sequence or at the terminal end of a sequence.Modifications can include derivatives as described elsewhere herein.

Peptides are cleared by the kidneys or phagocytes readily and shortlyafter administration. Moreover, peptides are susceptible to degradationby proteolytic enzymes. Linking of conotoxin peptides to fatty acylchains (lipidation) of different lengths and structures can increase thehalf-life of peptides in circulation by promoting interaction withproteins in the blood such as albumin, which act as carriers. Suitablelipidated moieties include fully saturated lipids as well as unsaturatedlipids such as mono-, bis-, tris-, and poly-unsaturated lipids. In someembodiments a core lipid moiety may be conjugated with more than oneconotoxin peptide. For example, two of the same conotoxin peptides maybe conjugated to a single lipid moiety.

An activated ester of a fatty acid, such as a N-Hydroxysuccinimidylester or other activated ester derived from a fatty acid with a freecarboxylic acid and a commercially available peptide coupling reagent,is mixed with the conotoxin peptide of interest that contains a freeamine such as N-terminal glycine in a solvent such as dimethylformamideand a base like diisopropylethylamine. The mixture is stirred in thedark for 12-16 hours and the lipidated conotoxin peptide product isisolated by semi-preparative reversed phase chromatography.

Modifications of RgIA analogs described herein also include fusion ofthe peptide to the Fc domain of IgG, thus combining the biologicalactivity of the RgIA peptides with the stability of monoclonalantibodies. As described herein, these RgIA peptibodies would begenerated by recombinant technology by fusing an RgIA analog in-framewith the Fc portion of human IgG. These peptide-Fc fusion proteinsgenerally have a molecular weight of less than 60-70 kDa, orapproximately half the weight of monoclonal antibodies. Incorporation ofthe Fc portion of IgG in peptibodies can prolong the half-life throughFcRn protection. Dimerization of two Fc regions increases the number ofactive peptides interacting with the target up to two-fold (Wu et al.,2014).

In certain embodiments, the peptide is fused to other domains of IgG orto albumin.

The presence of modified amino acids may be advantageous in, forexample, (a) increasing peptide serum half-life and/or functional invivo half-life, (b) reducing peptide immunogenicity, (c) increasingpeptide storage stability, (d) increasing peptide solubility, (e)prolonging circulating time, (f) increasing bioavailability, e.g.increasing the area under the curve (AUC_(sc)), and/or (g) increasedbuccal or oral bioavailability by increasing mucosal absorption. Aminoacid(s) can be modified, for example, co-translationally orpost-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means. The modified amino acid can be within thesequence or at the terminal end of a sequence. Modifications can includederivatives as described elsewhere herein.

The C-terminus may be a carboxylic acid or an amide group. The presentdisclosure also relates to the RgIA analogs further modified by (i)additions made to the C-terminus, such as Tyr, iodo-Tyr, a fluorescenttag, and/or (ii) additions made to the N-terminus, such as Tyr,iodo-Tyr, pyroglutamate, or a fluorescent tag.

In addition, residues or groups of residues known to the skilled artisanto improve stability can be added to the C-terminus and/or N-terminus.Also, residues or groups of residues known to the skilled artisan toimprove oral availability can be added to the C-terminus and/orN-terminus.

In certain embodiments, modification of the N-terminus includesacylation including N-formyl, N-acetyl, N-propyl, and long chain fattyacid groups. In certain embodiments modification of the N-terminusincludes addition of a PYRO group. In certain embodiments, modificationof the C-terminus and/or N-terminus includes fattylation by the additionof fatty acids 4 to 24, 10 to 18, or 12 to 16 carbon atoms in length.

In certain embodiments, modification of the peptide includes linkage ofthe peptide to fluorescent labels, including fluorescent dyes.

In certain embodiments, modification of the peptide includes replacementof one or more of the disulfide bonds with one or more of the following:dicarba bridges as alkane (via hydrogenation of alkene), Z-alkene,E-alkene, thioether, selenoether, trisulfide, tetrasulfide, polyethoxyether, aliphatic linkers, and/or a combination of aliphatic linker withone or more alkene moieties (Z- or E-isomers) that are synthesized viaring-closing metathesis reactions.

In certain embodiments, modification of the peptide includes PEGylation.PEGylation consists of the addition of one or more poly-(ethyleneglycol) (PEG) molecules to a peptide or protein, and often enhancesprotein and peptide delivery (Davies et al., 1977). Peptides are clearedby the kidneys phagocytes readily and shortly after administration.Moreover, peptides are susceptible to degradation by proteolytic enzymesin the blood. Linking of conopeptides to polyethyelen glycol (PEG) ofdifferent lengths and structures can increase the half-life of peptidesin circulation. PEGylation increases the molecular weight of the peptideand thus reduces the rate with which it is filtrated in the kidneys;PEGylation can also shield the peptide from proteases and macrophagesand other cells of the reticuloendothelial system (RES) that can removeit. In addition, PEGylation may reduce any immunogenicity associatedwith a foreign peptide.

An example of how conotoxin peptides can be conjugated to PEG isconjugation of a methoxy poly(ethylene glycol)-succinimidyl valerate toconotoxin peptide RgIA analog CSP-4-NH2 (SEQ ID NO:25). 5-10 mg ofconotoxin peptide and mPEG-butyraldehyde are reacted at a 1.5:1 molarratio by stirring in 0.25 mL of anhydrous dimethyl formamide in thepresence of 0.0026 mL N,N-diisopropylethylamine at room temperature for16 hours in the dark. Reaction completeness and the concentration ofPEGylated conotoxin peptide is measured by reverse phase chromatographyusing a Poroshell C18 column. In another type of PEG conjugationreaction, a methoxy poly(ethylene glycol) (i.e., PEG)-butyraldehyde isjoined to a conotoxin peptide. 5-10 mg of conotoxin peptide CSP-4-NH2and mPEG-butyraldehyde are reacted at a 1.5:1 molar ratio by stirring in0.2 mL of 100% methanol at room temperature for 15 minutes. An aqueoussolution of sodium cyanoborohydride to a final concentration of 1 mg/mL,followed by mixing 16 hours at room temperature in the dark. Reactioncompleteness and the concentration of PEGylated-conotoxin peptide ismeasured by reverse phase chromatography using a Poroshell C18 column.mPEG-conjugated conotoxin peptides are purified by removal of excessconotoxin peptide by centrifugation in a desalting column. Samples arecentrifuged at 1000×g for 2 minutes in a methanol-equilibrated Zeba Spindesalting column, (2 mL volume, 7,000 molecular weight cut-off,ThermoScientific). Reaction completeness and the concentration ofPEGylated conotoxin peptide in spun-through material is measured byreverse phase chromatography using a Poroshell C18 column.

The present disclosure is further directed to derivatives of thedisclosed RgIA analogs. Derivatives include RgIA analogs having cyclicpermutations in which the cyclic permutants retain the native bridgingpattern of native conotoxin peptide (Craik, et al. (2001)), e.g., acyclized conotoxin peptide having an amide cyclized backbone such thatthe conotoxin peptide has no free N- or C-terminus in which theconotoxin peptide includes the native disulfide bonds (U.S. Pat. No.7,312,195). In one embodiment, the cyclized conotoxin peptide includes alinear conotoxin peptide and a peptide linker, wherein the N- andC-termini of the linear conotoxin peptide are linked via the peptidelinker to form the amide cyclized peptide backbone. In some embodiments,the peptide linker includes amino acids selected from Gly, Ala andcombinations thereof.

Various cyclization methods can be applied to the RgIA analogs describedherein. The RgIA analogs described herein can be readily cyclized usingalanine bridges as described in, for example, in Clark, et al., 2013,and Clark, et al., 2012. Cyclizing RgIA analogs can improve their oralbioavailability and reduce the susceptibility to proteolysis, withoutaffecting the affinity of the RgIA analogs for their specific targets.Cyclization occurs between the N- and C-termini and disulfide bridgesbetween C1-C3 and C2-C4, respectively, where the GAAGAG cyclizationlinker can be of any length between 1 and 8 amino acids and can becomposed of any amino acid sequence. In certain embodiments, cyclizationis done using alternative linkers such as non-peptide linkers includingPolyethoxy ethers, aliphatic linkers, and/or any combination ofaliphatic linker with one or more alkene moieties (Z- or E-isomers) inthe hydrocarbon chain that can be synthesized via ring-closingmetathesis reactions.

TABLE 7 Cyclized sequences of RglA analogs Sequence SEQ ID NO.GCCSDPRCRX3RCRGAAGAG 13 GCCTDPRCX2X3QCRGAAGAG 14 GCCTDPRCX2X3QCRRRGAAGAG15 GCCTDPRCX2X3QCYGAAGAG 16 GX4CTDPRX4X2X3QCRGAAGAG 17GCCTDPRCRX3QCFGAAGAG 18 GCCTDPRCRX3QCYGAAGAG 19 GCCTDPRCRX3QCWGAAGAG 20X3 = des-X3, Ser, or Thr

Embodiments disclosed herein include the RgIA analogs described hereinas well as variants, D-substituted analogs, modifications, andderivatives of the RgIA analogs described herein. In some embodiments,variants, D-substituted analogs, modifications, and derivatives have 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 sequenceadditions, deletions, stop positions, substitutions, replacements,conjugations, associations, or permutations. Each conotoxin peptidedisclosed herein may also include additions, deletions, stop positions,substitutions, replacements, conjugations, associations, or permutationsat any position including positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, or 18 of an analog conotoxin peptide sequencedisclosed herein.

In some embodiments an Xaa position can be included in any position ofan analog conotoxin peptide, wherein Xaa represents an addition,deletion, stop position, substitution, replacement, conjugation,association or permutation. In particular embodiments, each analogconotoxin peptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18 Xaa positions at one or more of positions 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18.

An analog can have more than one change (addition, deletion, stopposition, substitution, replacement, conjugation, association, orpermutation) and qualify as one or more of a variant, D-substitutedanalog, modification, and/or derivative. That is, inclusion of oneclassification of analog, variant, D-substituted analog, modificationand/or derivative is not exclusive to inclusion in other classificationsand all are collectively referred to as “conotoxin peptides” herein.

The conotoxin peptides can be prepared using recombinant DNA technology.Conotoxin peptides may also be prepared using Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used. Solid-phase synthesis is commenced from the C-terminusof the conotoxin peptide by coupling a protected α-amino acid to asuitable resin. Such a starting material can be prepared by attaching anα-amino-protected amino acid by an ester linkage to a chloromethylatedresin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine(BHA) resin or para-methylbenzhydrylamine (MBHA) resin. Preparation ofthe hydroxymethyl resin is described by Bodansky et al. (1966).Chloromethylated resins are commercially available from Bio RadLaboratories (Richmond, Calif.). The preparation of such a resin isdescribed by Stewart and Young (1969). BHA and MBHA resin supports arecommercially available, and are generally used when the desiredconotoxin peptide being synthesized has an unsubstituted amide at theC-terminus. Thus, solid resin supports may be any of those known in theart, such as one having the formulae —O—CH2-resin support, —NH BHA resinsupport, or —NH-MBHA resin support. When the unsubstituted amide isdesired, use of a BHA or MBHA resin can be advantageous because cleavagedirectly gives the amide. In case the N-methyl amide is desired, it canbe generated from an N-methyl BHA resin. Should other substituted amidesbe desired, the teaching of U.S. Pat. No. 4,569,967 can be used, orshould still other groups than the free acid be desired at theC-terminus, it is possible to synthesize the conotoxin peptide usingclassical methods as set forth in the Houben-Weyl text (1974).

The C-terminal amino acid, protected by Boc or Fmoc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in Horiki etal. (1978), using KF in dimethylformamide (DMF) at about 60° C. for 24hours with stirring, when a conotoxin peptide having free acid at theC-terminus is to be synthesized. Following the coupling of theBOC-protected amino acid to the resin support, the α-amino protectinggroup can be removed, as by using trifluoroacetic acid (TFA) inmethylene chloride or TFA alone. The deprotection can be carried out ata temperature between 0° C. and room temperature. Other standardcleaving reagents, such as HCl in dioxane, and conditions for removal ofspecific α-amino protecting groups may be used as described in Schroder& Lubke (1965).

After removal of the α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids can be coupled step-wise in thedesired order to obtain an intermediate compound or as an alternative toadding each amino acid separately in the synthesis, some of them may becoupled to one another prior to addition to the solid phase reactor.Selection of an appropriate coupling reagent is within the skill of theart. Illustrative coupling reagents includeN,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU in thepresence of HoBt or HoAt).

The activating reagents used in the solid phase synthesis of peptidesincluding conotoxin peptides are well known in the art. Examples ofsuitable activating reagents include carbodiimides, such asN,N′-diisopropylcarbodiimide and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptidecoupling are described by Schroder & Lubke (1965) and Kapoor (1970).

Each protected amino acid or amino acid sequence can be introduced intothe solid-phase reactor in a twofold or more excess, and the couplingmay be carried out in a medium of DMF:CH2Cl2 (1:1) or in DMF or CH2Cl2alone. In cases where intermediate coupling occurs, the couplingprocedure can be repeated before removal of the α-amino protecting groupprior to the coupling of the next amino acid. The success of thecoupling reaction at each stage of the synthesis, if performed manually,can be monitored by the ninhydrin reaction, as described by Kaiser, etal. (1970). Coupling reactions can be performed automatically, as on aBeckman 990 automatic synthesizer, using a program such as that reportedin Rivier, et al. (1978).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group can be first removed usingTFA/ethanedithiol prior to cleaving the peptide from the resin with HFto eliminate potential S-alkylation. When using hydrogen fluoride or TFAfor cleaving, one or more scavengers such as anisole, cresol, dimethylsulfide and methylethyl sulfide can be included in the reaction vessel.

Cyclization of the linear conotoxin peptide can be effected, as opposedto cyclizing the conotoxin peptide while a part of the peptido-resin, tocreate bonds between Cys residues. To effect such a disulfide cyclizinglinkage, a fully protected conotoxin peptide can be cleaved from ahydroxymethylated resin or a chloromethylated resin support byammonolysis, as is well known in the art, to yield the fully protectedamide intermediate, which is thereafter suitably cyclized anddeprotected. Alternatively, deprotection, as well as cleavage of theconotoxin peptide from the above resins or a benzhydrylamine (BHA) resinor a methylbenzhydrylamine (MBHA), can take place at 0° C. withhydrofluoric acid (HF) or TFA, followed by oxidation as described above.

The conotoxin peptides can also be synthesized using an automaticsynthesizer. In these embodiments, amino acids can be sequentiallycoupled to an MBHA Rink resin (typically 100 mg of resin) beginning atthe C-terminus using an Advanced Chemtech 357 Automatic PeptideSynthesizer. Couplings are carried out using 1,3-diisopropylcarbodimidein N-methylpyrrolidinone (NMP) or by2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) and diethylisopropylethylamine (DIEA). The Fmoc protecting groupcan be removed by treatment with a 20% solution of piperidine indimethylformamide (DMF). Resins are subsequently washed with DMF(twice), followed by methanol and NMP.

Conotoxin peptides can be formulated within pharmaceutical compositions.“Pharmaceutical compositions” mean physically discrete coherent unitssuitable for medical administration. “Pharmaceutical composition indosage unit form” means physically discrete coherent units suitable formedical administration, each containing a therapeutically effectiveamount, or a multiple (up to four times) or sub-multiple (down to afortieth) of a therapeutically effective amount of a conotoxin peptidewith a pharmaceutically acceptable carrier. Whether the pharmaceuticalcomposition contains a daily dose, or for example, a half, a third or aquarter of a daily dose, will depend on whether the pharmaceuticalcomposition is to be administered once or, for example, twice, threetimes, or four times a day, respectively.

The amount and concentration of a conotoxin peptide in a pharmaceuticalcomposition, as well as the quantity of the pharmaceutical compositioncan be selected based on clinically relevant factors, the solubility ofthe conotoxin peptide in the pharmaceutical composition, the potency andactivity of the conotoxin peptide, and the manner of administration ofthe pharmaceutical composition. It is only necessary that the conotoxinpeptide constitute a therapeutically effective amount, i.e., such that asuitable effective dosage will be consistent with the dosage formemployed in single or multiple unit doses.

The pharmaceutical compositions will generally contain from 0.0001 to 99wt. %, preferably 0.001 to 50 wt. % or from 0.01 to 10 wt. % of theconotoxin peptide by weight of the total composition. In addition to theconotoxin peptide, the pharmaceutical compositions can also containother drugs or agents. Examples of other drugs or agents includeanalgesic agents, cytokines, and therapeutic agents in all of the majorareas of clinical medicine. When used with other drugs or agents, theconotoxin peptides may be delivered in the form of drug cocktails. Acocktail is a mixture of any one of the conotoxin peptides with anotherdrug or agent. In this embodiment, a common administration vehicle(e.g., pill, tablet, implant, pump, injectable solution, etc.) wouldcontain both the conotoxin peptide in combination with the other drugsor agents. The individual components of the cocktail can each beadministered in therapeutically effective amounts or theiradministration in combination can create a therapeutically effectiveamount.

Pharmaceutical compositions include pharmaceutically acceptable carriersincluding those that do not produce significantly adverse, allergic, orother untoward reactions that outweigh the benefit of administration,whether for research, prophylactic, and/or therapeutic treatments.Illustrative pharmaceutically acceptable carriers and formulations aredisclosed in Remington, 2005. Moreover, pharmaceutical compositions canbe prepared to meet sterility, pyrogenicity, and/or general safety andpurity standards as required by U.S. Food and Drug Administration (FDA)Office of Biological Standards, and/or other relevant regulatoryagencies.

Typically, a conotoxin peptide will be admixed with one or morepharmaceutically acceptable carriers chosen for the selected mode ofadministration. For examples of delivery methods see U.S. Pat. No.5,844,077.

Illustrative generally used pharmaceutically acceptable carriers includeany and all bulking agents, fillers, solvents, co-solvents, dispersionmedia, coatings, surfactants, antioxidants, preservatives, isotonicagents, releasing agents, absorption delaying agents, salts,stabilizers, buffering agents, chelating agents, gels, binders,disintegration agents, wetting agents, emulsifiers, lubricants, coloringagents, flavoring agents, sweetening agents, and perfuming agents.

Illustrative buffering agents include citrate buffers, succinatebuffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalatebuffers, lactate buffers, acetate buffers, phosphate buffers, histidinebuffers, and trimethylamine salts.

Illustrative preservatives include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides, hexamethonium chloride, alkyl parabens,methyl paraben, propyl paraben, catechol, resorcinol, cyclohexanol, and3-pentanol.

Illustrative isotonic agents include polyhydric sugar alcohols,trihydric sugar alcohols, or higher sugar alcohols, such as glycerin,erythritol, arabitol, xylitol, sorbitol, and mannitol.

Illustrative stabilizers include organic sugars, polyhydric sugaralcohols, polyethylene glycol, sulfur-containing reducing agents, aminoacids, low molecular weight peptides, immunoglobulins, hydrophilicpolymers, and polysaccharides.

Illustrative antioxidants include ascorbic acid, methionine, vitamin E,cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodiumsulfite, oil soluble antioxidants, ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, metal chelating agents, citric acid,ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, andphosphoric acid.

Illustrative lubricants include sodium lauryl sulfate and magnesiumstearate.

Illustrative pharmaceutically acceptable salts include acidic and/orbasic salts, formed with inorganic or organic acids and/or bases,preferably basic salts. While pharmaceutically acceptable salts arepreferred, particularly when employing the conotoxin peptides asmedicaments, other salts find utility, for example, in processing theseconotoxin peptides, or where non-medicament-type uses are contemplated.Salts of these conotoxin peptides may be prepared by techniquesrecognized in the art.

Illustrative pharmaceutically acceptable salts include inorganic andorganic addition salts, such as hydrochloride, sulphates, nitrates,phosphates, acetates, trifluoroacetates, propionates, succinates,benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates,isothionates, theophylline acetates, and salicylates. Lower alkylquaternary ammonium salts can also be used.

For oral administration, the conotoxin peptides can be formulated intosolid or liquid preparations such as capsules, pills, tablets, lozenges,melts, powders, suspensions, or emulsions. In preparing the compositionsin oral dosage form, any of the usual pharmaceutically acceptablecarriers may be employed, such as, for example, carriers such asstarches, sugars, diluents, granulating agents, lubricants, binders,disintegrating agents, and the like in the case of oral solidpreparations (such as, for example, powders, capsules and tablets); orwater, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, suspending agents, and the like in the case of oralliquid preparations (such as, for example, suspensions, elixirs andsolutions). Because of their ease in administration, tablets andcapsules can represent an advantageous oral dosage unit form, in whichcase solid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar-coated or enteric-coated by standard techniques.The conotoxin peptide can be encapsulated to make it stable to passagethrough the gastrointestinal tract while at the same time, in certainembodiments, allowing for passage across the blood brain barrier. Seefor example, WO 96/11698.

For parenteral administration, the conotoxin peptides may be dissolvedin a pharmaceutically acceptable carrier and administered as either asolution or a suspension. Illustrative pharmaceutically acceptablecarriers include water, saline, dextrose solutions, fructose solutions,ethanol, or oils of animal, vegetative, or synthetic origin. The carriermay also contain other ingredients, for example, preservatives,suspending agents, solubilizing agents, buffers, and the like.

The conotoxin peptides can be in powder form for reconstitution in theappropriate pharmaceutically acceptable carrier at the time of delivery.In another embodiment, the unit dosage form of the conotoxin peptide canbe a solution of the conotoxin peptide, or a pharmaceutically acceptablesalt thereof, in a suitable diluent in sterile, hermetically sealedampoules or sterile syringes.

Conotoxin peptides can also be formulated as depot preparations. Depotpreparations can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salts.

Additionally, conotoxin peptides can be formulated as sustained-releasesystems utilizing semipermeable matrices of solid polymers containing atleast one compound. Various sustained-release materials have beenestablished and are well known by those of ordinary skill in the art.Sustained-release systems may, depending on their chemical nature,release conotoxin peptides following administration for a few weeks upto over 100 days.

Administration of the conotoxin peptide can also be achieved using pumps(see, e.g., Luer et al., (1993), Zimm, et al. (1984) and Ettinger, etal. (1978)); microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883,4,353,888, and 5,084,350); continuous release polymer implants (see,e.g., U.S. Pat. No. 4,883,666); and macroencapsulation (see, e.g., U.S.Pat. Nos. 5,284,761, 5,158,881, 4,976,859, and 4,968,733 and publishedPCT patent applications WO92/19195, WO 95/05452);

When the conotoxin peptides are administered intrathecally, they mayalso be dissolved in cerebrospinal fluid. Naked or unencapsulated cellgrafts to the CNS can also be used. See, e.g., U.S. Pat. Nos. 5,082,670and 5,618,531.

The conotoxin peptides of the present disclosure, and pharmaceuticalcompositions thereof, are useful in methods of treating conditionsassociated with the α9α10 receptor subtype of the nicotinicacetylcholine receptor (nAChR) in a subject. The activity of certainα-conotoxins, including RgIA and its analogs, in blocking the α9α10subtype of nAChR has been shown herein in studies using oocytes thatexpress different subtypes of the nAChR (Ellison et al., 2006; Vincleret al., 2006; WO 2008/011006; US 2009/0203616; US 2012/0220539). Theactivity of α-conotoxins, including RgIA, as an antinociceptive and ananalgesic has been shown in studies of chronic constriction injury(Vincler, et al., 2006; WO 2008/011006; US 2009/0203616). The activityof α-conotoxins, including RgIA, in inhibiting migration of immune cellshas been shown in studies of chronic constriction injury (Vincler, etal., 2006; WO 2008/011006; US 2009/0203616).

Methods described herein include administering to a subject in needthereof a therapeutically effective amount of a disclosed conotoxinpeptide or a pharmaceutically acceptable salt thereof, wherein thedisclosed conotoxin peptide blocks the α9α10 subtype of the nAChR.Conotoxin peptides that block the α9α10 subtype of nAChR are useful fortreating pain, for treating inflammation and/or inflammatory conditionsand for treating cancers and/or cancer related pain. In certainembodiments, the conotoxin peptides are effective based on their abilityto inhibit the migration of immune cells. In other embodiments, thecompounds are effective based on their ability to slow demyelinationand/or increase the number of intact nerve fibers.

Methods disclosed herein include treating subjects (humans, veterinaryanimals (dogs, cats, reptiles, birds, etc.), livestock (horses, cattle,goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice,fish, etc.)) with conotoxin peptides disclosed herein includingpharmaceutically-acceptable salts and prodrugs thereof. Treatingsubjects includes delivering therapeutically effective amounts of thedisclosed conotoxin peptides. Therapeutically effective amounts includethose that provide effective amounts, prophylactic treatments, and/ortherapeutic treatments.

An “effective amount” is the amount of a conotoxin peptide necessary toresult in a desired physiological change in the subject. Effectiveamounts are often administered for research purposes. Effective amountsdisclosed herein result in a desired physiological change in a researchassay intended to study the effectiveness of a conotoxin peptide in thetreatment of pain, inflammatory conditions, inflammation, and/or cancer.

A “prophylactic treatment” includes a treatment administered to asubject who does not display signs or symptoms of pain, an inflammatorycondition, inflammation, and/or cancer or a subject who displays onlyearly signs or symptoms of pain, an inflammatory condition,inflammation, and/or cancer such that treatment is administered for thepurpose of diminishing, preventing, or decreasing the risk of developingthe pain, inflammatory condition, inflammation, and/or cancer further.Thus, a prophylactic treatment functions as a preventative treatmentagainst pain, an inflammatory condition, inflammation, and/or cancer.

A “therapeutic treatment” includes a treatment administered to a subjectwho displays symptoms or signs of pain, an inflammatory condition,inflammation, and/or cancer and is administered to the subject for thepurpose of diminishing or eliminating those signs or symptoms of thepain, inflammatory condition, inflammation, and/or cancer. Thetherapeutic treatment can reduce, control, or eliminate the presence oractivity of pain, an inflammatory condition, inflammation, and/or cancerand/or reduce control or eliminate side effects of pain, an inflammatorycondition, inflammation, and/or cancer.

Illustrative types of pain that can be treated include general pain,chronic pain, neuropathic pain, nociceptive pain, and inflammatory pain.In addition, these types of pain can be associated with and/or inducedby causes including: peripheral nerve or nociceptor damage, inflammatoryconditions, metabolic disorders, virus infection, cancers, pain inducedby chemotherapeutic agents, pain induced after surgical procedure, andpain induced by burn or other physical tissue injury.

Therapeutically effective amounts in the treatment ofchemotherapy-induced neuropathic pain (CINP) can include those thatdecrease mechanical hyperalgesia, mechanical allodynia (pain due to astimulus that does not normally cause pain), thermal (heat-induced)hyperalgesia, thermal (cold-induced) allodynia, the number of migratingimmune cells, levels of inflammatory mediators, and/or subject-reportedsubjective pain levels.

Therapeutically effective amounts in the treatment of burn-inducedneuropathic pain can include those that decrease mechanicalhyperalgesia, mechanical allodynia, thermal (heat-induced) hyperalgesia,thermal (cold-induced) allodynia, the number of migrating immune cells,levels of inflammatory mediators, and/or subject-reported subjectivepain levels.

Therapeutically effective amounts in the treatment of post-operativeneuropathic pain can include those that decrease mechanicalhyperalgesia, mechanical allodynia, thermal (heat-induced) hyperalgesia,thermal (cold-induced) allodynia, the number of migrating immune cells,levels of inflammatory mediators, and/or subject-reported subjectivepain levels.

Illustrative inflammatory conditions that can be treated includeinflammation, chronic inflammation, rheumatic diseases (includingarthritis, lupus, ankylosing spondylitis, fibromyalgia, tendonitis,bursitis, scleroderma, and gout), sepsis, fibromyalgia, inflammatorybowel disease (including ulcerative colitis and Crohn's disease),sarcoidosis, endometriosis, uterine fibroids, inflammatory skin diseases(including psoriasis and impaired wound healing), inflammatoryconditions of the lungs (including asthma and chronic obstructivepulmonary disease), diseases associated with inflammation of the nervoussystem (including multiple sclerosis, Parkinson's Disease andAlzheimer's Disease), periodontal disease, and cardiovascular disease.

Therapeutically effective amounts in the treatment of inflammatoryconditions can include those that decrease levels of inflammatorymarkers at the gene expression or protein level and/or reduce the numberof migrating immune cells. In addition, pain associated withinflammatory conditions can be treated by therapeutically effectiveamounts that result in the decrease of mechanical hyperalgesia,mechanical allodynia, thermal (heat-induced) hyperalgesia, thermal(cold-induced) allodynia, and/or subject-reported subjective painlevels.

Illustrative cancers that can be treated include breast cancers.α9-nAChR is overexpressed in human breast tumor tissue (Lee et al.,2010a) and receptor inhibition by siRNA or other mechanism reduced invitro and in vivo carcinogenic properties of breast cancer cells,including inhibition of cancer cell proliferation (Chen et al., 2011).In certain embodiments, RgIA analogs are used in therapeutic amounts inorder to inhibit tumor growth by inhibition of α9-nAChR.

Therapeutically effective amounts in the treatment of cancers, such asbreast cancers, can include those that decrease a number of tumor cells,decrease the number of metastases, decrease tumor volume, increase lifeexpectancy, induce apoptosis of cancer cells, induce cancer cell death,induce chemo- or radiosensitivity in cancer cells, inhibit angiogenesisnear cancer cells, inhibit cancer cell proliferation cells, inhibittumor growth cells, prevent metastasis, prolong a subject's life, reducecancer-associated pain, and/or reduce relapse or re-occurrence of thecancer in a subject following treatment.

For administration, therapeutically effective amounts can be initiallyestimated based on results from in vitro assays and/or animal modelstudies. For example, a dose can be formulated in animal models toachieve a circulating concentration range that includes an IC50 asdetermined in cell culture against a particular target. Such informationcan be used to more accurately determine therapeutically effectiveamounts in subjects of interest.

The actual amount administered to a particular subject as atherapeutically effective amount can be determined by a physician,veterinarian, or researcher taking into account parameters such asphysical and physiological factors including target; body weight;severity of condition; type of pain, inflammatory condition, or cancer;previous or concurrent therapeutic interventions; idiopathy of thesubject; and route of administration.

Dosage may be adjusted appropriately to achieve desired conotoxinpeptide levels, locally or systemically. Typically the conotoxinpeptides of the present disclosure exhibit their effect at a dosagerange from 0.001 mg/kg to 250 mg/kg, preferably from 0.01 mg/kg to 100mg/kg of the conotoxin peptide, more preferably from 0.05 mg/kg to 75mg/kg. A suitable dose can be administered in multiple sub-doses perday. Typically, a dose or sub-dose may contain from 0.1 mg to 500 mg ofthe conotoxin peptide per unit dosage form. A more preferred dosage willcontain from 0.5 mg to 100 mg of conotoxin peptide per unit dosage form.

Additional doses which are therapeutically effective amounts can oftenrange from 0.1 to 5 μg/kg or from 0.5 to 1 μg/kg. In other examples, adose can include 1 μg/kg, 5 μg/kg, 10 μg/kg, 15 μg/kg, 20 μg/kg, 25μg/kg, 30 μg/kg, 35 μg/kg, 40 μg/kg, 45 μg/kg, 50 μg/kg, 55 μg/kg, 60μg/kg, 65 μg/kg, 70 μg/kg, 75 μg/kg, 80 μg/kg, 85 μg/kg, 90 μg/kg, 95μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg, 250 μg/kg, 350 μg/kg, 400 μg/kg,450 μg/kg, 500 μg/kg, 550 μg/kg, 600 μg/kg, 650 μg/kg, 700 μg/kg, 750μg/kg, 800 μg/kg, 850 μg/kg, 900 μg/kg, 950 μg/kg, 1000 μg/kg, 0.1 to 5mg/kg, or from 0.5 to 1 mg/kg. In other examples, a dose can include 1mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 150mg/kg, 200 mg/kg, 250 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg,550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg, or more.

In particular embodiments, dosages can be initiated at lower levels andincreased until desired effects are achieved. In the event that theresponse in a subject is insufficient at such doses, even higher doses(or effective higher doses by a different, more localized deliveryroute) may be employed to the extent that subject tolerance permits.Continuous dosing over, for example, 24 hours, or multiple doses per dayare contemplated to achieve appropriate systemic levels of conotoxinpeptide.

Therapeutically effective amounts can be achieved by administeringsingle or multiple doses during the course of a treatment regimen (e.g.,daily, every other day, every 3 days, every 4 days, every 5 days, every6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months,every 3 months, every 4 months, every 5 months, every 6 months, every 7months, every 8 months, every 9 months, every 10 months, every 11months, or yearly.

A variety of administration routes are available. The particular modeselected can depend upon the particular conotoxin peptide delivered, theseverity of pain, inflammatory condition or cancer being treated, andthe dosage required to provide a therapeutically effective amount. Anymode of administration that is medically acceptable, meaning any modethat provides a therapeutically effective amount of the conotoxinpeptide without causing clinically unacceptable adverse effects thatoutweigh the benefits of administration according to sound medicaljudgment, can be used. Illustrative routes of administration includeintravenous, intradermal, intraarterial, intraparenteral, intranasal,intranodal, intralymphatic, intraperitoneal, intralesional,intraprostatic, intravaginal, intrarectal, topical, intrathecal,intratumoral, intramuscular, intravesicular, oral, subcutaneous, and/orsublingual administration and more particularly by intravenous,intradermal, intraarterial, intraparenteral, intranasal, intranodal,intralymphatic, intraperitoneal, intralesional, intraprostatic,intravaginal, intrarectal, topical, intrathecal, intratumoral,intramuscular, intravesicular, oral, subcutaneous, and/or sublingualinjection.

In one embodiment, the conotoxin peptide is delivered directly into thecentral nervous system (CNS), preferably to the brain ventricles, brainparenchyma, the intrathecal space, or other suitable CNS location.

Alternatively, targeting therapies may be used to deliver the conotoxinpeptide more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands.

Conotoxin peptides can also be administered in a cell based deliverysystem in which a nucleic acid sequence encoding the conotoxin peptideis introduced into cells designed for implantation in the body of thesubject. In particular embodiments, this delivery method can be used inthe spinal cord region. Suitable delivery systems are described in U.S.Pat. No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO96/40871, WO 96/40959, and WO 97/12635.

Suitable nucleic acid sequences can be prepared synthetically for eachconotoxin peptide on the basis of the disclosed sequences and the knowngenetic code. In some embodiments, the polynucleotide includes aplasmid, a cDNA, or an mRNA that can include, e.g., a sequence (e.g., agene) for expressing a conotoxin peptide. Suitable plasmids includestandard plasmid vectors and minicircle plasmids that can be used totransfer a gene to a cell. The polynucleotides (e.g., minicircleplasmids) can further include any additional sequence information tofacilitate transfer of the genetic material (e.g., a sequence encoding aconotoxin peptide) to a cell. For example, the polynucleotides caninclude promoters, such as general promoters, tissue-specific promoters,cell-specific promoters, and/or promoters specific for the nucleus orcytoplasm. Promoters and plasmids (e.g., minicircle plasmids) aregenerally well known in the art and can be prepared using conventionaltechniques. As described further herein, the polynucleotides can be usedto transfect cells. Unless otherwise specified, the terms transfect,transfected, or transfecting can be used to indicate the presence ofexogenous polynucleotides or the expressed polypeptide therefrom in acell. A number of vectors are known to be capable of mediating transferof genes to cells, as is known in the art.

Briefly, the term “gene” refers to a nucleic acid sequence that encodesa conotoxin peptide. This definition includes various sequencepolymorphisms, mutations, and/or sequence variants wherein suchalterations do not affect the function of the encoded conotoxin peptide.The term “gene” may include not only coding sequences but alsoregulatory regions such as promoters, enhancers, and terminationregions. “Gene” further can include all introns and other DNA sequencesspliced from the mRNA transcript, along with variants resulting fromalternative splice sites. Nucleic acid sequences encoding the conotoxinpeptide can be DNA or RNA that directs the expression of the conotoxinpeptide. These nucleic acid sequences may be a DNA strand sequence thatis transcribed into RNA or an RNA sequence that is translated intoprotein. The nucleic acid sequences include both the full-length nucleicacid sequences as well as non-full-length sequences derived from thefull-length protein. The sequences can also include degenerate codons ofthe native sequence or sequences that may be introduced to provide codonpreference in a specific cell type. Gene sequences to encode conotoxinpeptide disclosed herein are available in publicly available databasesand publications.

As stated, conotoxin peptides disclosed herein block the α9α10 subtypeof the nAChR. Blocking can be measured by any effective means. In oneembodiment, blocking is measured as the displacement of labeled RgIAfrom the α9α10 subtype of the nAChR by a conotoxin peptide disclosedherein. In one embodiment, blocking can be a 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% displacement of labeled RgIA from theα9α10 subtype of the nAChR by a conotoxin peptide disclosed herein.

In a second embodiment, blocking can be measured by conducting abiological assay on a conotoxin peptide disclosed herein to determineits therapeutic activity as compared to the results obtained from thebiological assay of RgIA. In one embodiment, blocking can be 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% greater therapeuticactivity of conotoxin peptide disclosed herein when compared to RgIA asmeasured by the biological assay.

In a third embodiment, the binding affinity of a conotoxin peptidedisclosed herein to the α9α10 subtype of the nAChR can be measured andcompared to the binding affinity of RgIA to the α9α10 subtype of thenAChR. In one embodiment, blocking can be a 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% greater binding affinity of theconotoxin peptide disclosed herein over RgIA.

In a fourth embodiment, the effect of a conotoxin peptide disclosedherein on the function of the α9α10 subtype of the nAChR is analyzed bymeasuring the effect in functional assays, such as electrophysiologicalassays, calcium imaging assays, and the like. In one embodiment,blocking includes a 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% reduction in the function of the α9α10 subtype of the nAChR asmeasured by a functional assay when compared to RgIA.

Conotoxin peptides disclosed herein are also useful in methods ofidentifying drug candidates for use in treating conditions associatedwith the α9α10 subtype of the nAChR. These methods include screening adrug candidate for its ability to block the activity of the α9α10subtype of the nAChR.

“Drug candidate” refers to any peptide (including antibodies or antibodyfragments) or compound (small molecule or otherwise) that may block orotherwise interfere with the activity of a target (i.e. the α9α10subtype). Small molecules may belong to any chemical class suspected tointeract with a peptide complex and expected to be pharmaceuticallyacceptable. Drug candidates can be found in nature, synthesized bycombinatorial chemistry approaches, and/or created via rational drugdesign.

Blocking can be measured as described elsewhere herein except that thedrug candidate can be compared to conotoxin peptides disclosed hereinrather than or in addition to RgIA. Conotoxin peptides are useful inmethods of identifying drug candidates that mimic the therapeuticactivity of the conotoxin peptide. Such methods include the steps of:(a) conducting a biological assay on a drug candidate to determine itstherapeutic activity; and (b) comparing the results obtained from thebiological assay of the drug candidate to the results obtained from thebiological assay of a conotoxin peptides disclosed herein.

Drug candidates may also interfere with the activity of the α9α10subtype through interaction with polynucleotides (e.g. DNA and/or RNA),and/or enzymes. Such drug candidates can be known or potential DNAmodifying agents, including DNA damaging agents (e.g. intercalatingagents that interfere with the structure of nucleic acids); DNA bindingagents; mismatch binding proteins; and/or alkylating agents.

One goal of rational drug design is to identify drug candidates whichare, for example, more active or stable forms of the conotoxin peptide,or which, e.g., enhance or interfere with the function of a peptide invivo. Several approaches for use in rational drug design includeanalysis of three-dimensional structure, alanine scans, molecularmodeling, and use of anti-id antibodies. Such techniques may includeproviding atomic coordinates defining a three-dimensional structure of aprotein complex formed by the conotoxin peptide and the α9α10 subtype ofthe nAChR, and designing or selecting drug candidates capable ofinterfering with the interaction between a conotoxin peptide and theα9α10 subtype of the nAChR based on the atomic coordinates.

Once a drug candidate is selected for further study or development, itsstructure can be modeled according to its physical properties, e.g.,stereochemistry, bonding, size, and/or charge, using data from a rangeof sources, e.g., spectroscopic techniques, x-ray diffraction data, andNMR. Computational analysis, similarity mapping (which models the chargeand/or volume of a drug candidate, rather than the bonding betweenatoms), and other techniques can be used in this modeling process.

When a drug candidate is selected, attachment of further chemical groupscan be evaluated. Chemical groups can be selected so that the drugcandidate is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while, in some embodiments,retaining or improving the biological activity of a lead conotoxinpeptide. Alternatively, where the drug candidate is peptide-based,further stability can be achieved by cyclizing the peptide, whichincreases its rigidity. The drug candidates with attached chemicalgroups can be further screened to see ensure they retain targetproperties. Further optimization or modification can then be carried outto arrive at one or more final drug candidates for in vivo or clinicaltesting.

Following selection and optimization of a drug candidate, the selectedand optimized drug candidate may be manufactured and/or used in apharmaceutical composition for administration to subjects.

The Examples below are included to demonstrate particular embodiments.Those of ordinary skill in the art should recognize in light of thepresent disclosure that many changes can be made to the specificembodiments disclosed herein and still obtain a like or similar resultwithout departing from the spirit and scope of the disclosure.

EXAMPLES Example 1. Amidation of the C-Terminus Increases the Stabilityof RgIA Analogs

The replacement of the hydroxyl group in the carboxyl group of theC-terminus of peptides by an amide group was done for two RgIA analogsto increase stability. CSP-2 (SEQ ID NO:3) was considerably more stable,as evidenced by the higher percentage of the peptide remaining, in abiological matrix when the C-terminus was amidated (i.e., addition ofNH2 to the C-terminus) compared to the original carboxyl group (FIG.2A). A similar finding was made with CSP-4 (SEQ ID NO:5) and shown inFIG. 2B.

Selected illustrative peptide sequences with amidation of the C-terminusto increase stability are described in Table 8.

TABLE 8 Peptide sequences with amidation of C-terminus Sequence SEQ IDNO. GCCSDPRCRYRCR-amide 21 GCCSDPRCRX12RCR-amide 22GCCTDPRCX11X12QCR-amide 23 GCCTDPRCX11X12QCRRR-amide 24GX28X28TDPRX28X11X12QX28Y-amide 25 GX13CTDPRX13X11X12QCR-amide 26GCCTDPRCRX12QCF-amide 27 GCCTDPRCRX12QCY-amide 28 GCCTDPRCRX12QCW-amide29 X11 = Citrulline X12 = 3-iodo-Tyrosine X13 = Selenocysteine X28= Cys, any natural amino acid, or any unnatural amino acid

Example 2. Lipidation of Conotoxin Peptides

Lipidated-succinimidyl valerate was conjugated CSP-4-NH2 (SEQ ID NO:25).5-10 mg of conotoxin peptide and lipidated succinimidyl valerate werereacted at a 1.5:1 molecular weight ratio by stirring in 0.25 mL ofanhydrous dimethyl formamide in the presence 0.0026 mLN,N-diisopropylethylamine at room temperature for 16 hours in the dark.Reaction completeness and the concentration of lipidated conotoxinpeptide is measured by reverse phase chromatography using a PoroshellC18 column. Lipidated conotoxin peptide in dimethyl formamide ispurified by reverse phase chromatography over a Hypersep C18 column witha gravity feed. The sample is loaded onto a calibrated column in 95%H₂O/5% methanol/0.1% formic acid. The column is loaded in the samebuffer. The sample is eluted in four bed-volume fractions of 95%methanol/5% H₂O/0.1% formic acid. Fractions shown to contain lipidatedconotoxin peptide by reverse phase chromatography using a Poroshell C18column are pooled, lyophilized, and resuspended in methanol.

FIG. 6 shows the pharmacokinetic and pharmacodynamic properties ofpeptide drugs increased by lipidation. FIG. 6 shows an increased inconcentration of CSP-4-NH2 when conjugated to a 12 or 16 carbon lipid.Stability of CSP-4-NH2 is also shown in FIG. 2B. Lipidation of CSP-4-NH2by an activated ester of a 12 carbon fatty acid creates C12-CSP-4-NH2and lipidation by an activated ester of a 16 carbon fatty acid createsC16-CSP-4-NH2. C12-CSP-4-NH2 could be detected for up 16 h, whileC16-CSP-4-NH2 could be detected for up to 24 h.

Example 3. Evaluation of Lipidated CSP-4 in Capsaicin Model

The capsaicin model of neuropathic pain was used to evaluate thetherapeutic potential of RgIA analogs to treat neuropathic pain. In thismodel, 30 μg of Capsaicin were injected intraplantarly in the rathindpaw to create capsaicin-induced pain in the rats. Thermalhyperalgesia as measured by the Hargreaves test (a measure ofsensitivity to pain; Hargreaves, et al., 1988) was performed at 15, 30,and 45 min following capsaicin injection. Paw withdrawal latency wasmeasured prior to capsaicin injection (Baseline). C12-CSP-4-NH2,C16-CSP-4-NH2, or vehicle without peptide was subcutaneously injected2-3 hours before the capsaicin injection. As can be seen in FIGS. 7A and7B, injection of lipidated C12-CSP-4-NH2 and C16-CSP-4-NH2 resulted inreduction of capsaicin-induced thermal hyperalgesia.

Example 4. Evaluation of Lipidated and PEGylated CSP-4 in CINP Model

CINP was induced in rats via intravenous injection of the platinum saltoxaliplatin (2.4 mg/kg) twice a week during 3 weeks. Mechanicalhyperalgesia is commonly induced in the CINP model by day 14 in whichthe therapeutic regimen initiates. Mechanical hyperalgesia was assessedusing the Randall-Selitto test. The Randall-Selitto test is a measure ofsensitivity to pain. As seen in FIGS. 8 and 9, lipidation andPEGylation, respectively, resulted in a reduction in hyperalgesia inthis rat model of neuropathic pain. Lipidation of CSP-4-NH2 (FIG. 8)with an activated ester of dodecanoic acid to create C12-CSP-4-NH2provided a therapeutic benefit that lasted 29 h. PEGylation of CSP-4-NH2(FIG. 9) with PEG-SVA to create PEG-SVA-CSP-4-NH2 extended thispharmacological therapeutic effect to over 3 days.

EXEMPLARY EMBODIMENTS Embodiment 1

A conotoxin peptide comprising the formula of SEQ ID NO:10.

Embodiment 2

A conotoxin peptide of embodiment 1, comprising the formula of SEQ IDNO:11.

Embodiment 3

A conotoxin peptide of embodiment 2, comprising the formula of SEQ IDNO:12.

Embodiment 4

A conotoxin peptide comprising the formula of any one from: SEQ IDNO:13-20.

Embodiment 5

A conotoxin peptide comprising the formula of any one from: SEQ ID NO:174-185.

Embodiment 6

A conotoxin peptide of any of embodiments 1-5, wherein the C-terminus ofthe peptide is an amide group (—NH2).

Embodiment 7

A conotoxin peptide of any of embodiments 1-6, wherein the peptide islinked to a fatty acid.

Embodiment 8

A conotoxin peptide of embodiment 7, wherein the fatty acid is a 3 to 60carbon fatty acid.

Embodiment 9

A conotoxin peptide of any of embodiments 1-8, wherein the amino acid atthe C terminus of the conotoxin peptide is replaced by the D-amino acidstereoisomer.

Embodiment 10

A conotoxin peptide of any of embodiments 1-9, wherein the N-terminalamino acid is an acetylated amino acid.

Embodiment 11

A conotoxin peptide of any of embodiments 1-10, wherein the peptide isbiotinylated.

Embodiment 12

A conotoxin peptide of any of embodiments 1-11, wherein the peptide ismethylated.

Embodiment 13

A conotoxin peptide of any of embodiments 1-12, wherein the peptide isphosphorylated at one or more sites.

Embodiment 14

A conotoxin peptide of any of embodiments 1-13, wherein the peptide isglycosylated.

Embodiment 15

A conotoxin peptide of any of embodiments 1-14, wherein the peptide islinked to a fluorescent dye or a fluorescent protein.

Embodiment 16

A conotoxin peptide of any of embodiments 1-15, wherein two cysteineresidues are each replaced with a natural or unnatural amino acid thatare then coupled for bridge formation.

Embodiment 17

A conotoxin peptide of embodiment 16, wherein each of the cysteineresidues is replaced with an (R)- or (S)-version of a naturallyoccurring amino acid selected from aspartic acid, glutamic acid orlysine.

Embodiment 18

A conotoxin peptide of embodiment 16, wherein a first of the twocysteine residues is replaced with an unnatural amino acid containingcarboxylic acid in a side chain and a second of the two cysteineresidues is replaced with an unnatural amino acid containing an aminegroup in a side chain.

Embodiment 19

A conotoxin peptide of embodiment 16, wherein each of the cysteineresidues is replaced with (S)-propargyl glycine or (S)-azidonorvaline.

Embodiment 20

A conotoxin peptide of any of embodiments 16-19, wherein the bridge is alactam bridge or a triazole bridge.

Embodiment 21

A conotoxin peptide of any of embodiments 1-14, wherein a linker isintroduced so as to generate an N-terminus to C-terminus cyclizedpeptide.

Embodiment 22

A conotoxin peptide of embodiment 21, wherein the linker consists of asequence of 1 to 100 amino acids.

Embodiment 23

A conotoxin peptide of embodiment 21, wherein the linker isnon-peptidic.

Embodiment 24

A conotoxin peptide of any of embodiments 1-14, wherein the peptide islinked to polyethylene glycol polymers.

Embodiment 25

A conotoxin peptide of any of embodiments 1-14, wherein the peptide isexpressed as a fusion to a protein.

Embodiment 26

A conotoxin peptide of embodiment 25, wherein the protein is the Fcportion of immunoglobulin G (IgG).

Embodiment 27

A pharmaceutical composition comprising the conotoxin peptide of any ofembodiments 1-26.

Embodiment 28

A pharmaceutically acceptable salt comprising the conotoxin peptide ofany of embodiments 1-26.

Embodiment 29

A method for treating at least one condition associated with the α9α10subtype of the nicotinic acetylcholine receptor (nAChR) in a subject inneed thereof comprising administering to the subject a therapeuticallyeffective amount of a conotoxin peptide, a composition comprising theconotoxin peptide, or a pharmaceutically acceptable salt comprising theconotoxin peptide, wherein the conotoxin peptide is the conotoxinpeptide of any of embodiments 1-26, thereby treating the condition.

Embodiment 30

A method of embodiment 29, wherein at least one condition is pain.

Embodiment 31

A method of embodiment 30, wherein the pain is general pain, chronicpain, neuropathic pain, nociceptive pain, inflammatory pain, paininduced by peripheral nerve damage, pain induced by an inflammatorydisorder, pain induced by a metabolic disorder, pain induced by cancer,pain induced by chemotherapy, pain induced by a surgical procedure,and/or pain induced by a burn.

Embodiment 32

A method of embodiment 31, wherein the pain is cancer-related chronicpain and/or cancer-related neuropathy.

Embodiment 33

A method of embodiment 29, wherein the at least one condition is aninflammatory condition.

Embodiment 34

A method of embodiment 33, wherein the inflammatory condition isinflammation, chronic inflammation, a rheumatic disease, sepsis,fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis,uterine fibroids, an inflammatory skin disease, an inflammatorycondition of the lungs, a disease associated with inflammation of thenervous system, periodontal disease, or cardiovascular disease.

Embodiment 35

A method of any of embodiments 33-34, wherein the inflammatory conditionis mediated by immune cells.

Embodiment 36

A method of any of embodiments 33-35, wherein the inflammatory conditionis long-term inflammation and peripheral neuropathy following injury.

Embodiment 37

A method of embodiment 29, wherein the at least one condition is painand inflammation.

Embodiment 38

A method of embodiment 29, wherein the at least one condition isinflammation and neuropathy.

Embodiment 39

A conotoxin peptide of any of embodiments 1-14, wherein the peptide bondbetween the aspartate residue and the proline residue in the Asp-Pro-Argregion is replaced by a non-peptidic bond in which a methylene group isincorporated between the carbonyl of aspartate and the nitrogen ofproline.

Embodiment 40

A conotoxin peptide of any of embodiments 1-14, wherein the aspartate inthe Asp-Pro-Arg region is replaced by amino malonic acid.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentinvention only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for the fundamentalunderstanding of the invention, the description taken with the drawingsand/or examples making apparent to those skilled in the art how theseveral forms of the invention may be embodied in practice.

The practice of the present disclosure employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture, and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1982); Sambrook et al.,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989); Sambrook and Russell, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,2001); Ausubel et al., Current Protocols in Molecular Biology (JohnWiley & Sons, updated through 2005); Glover, DNA Cloning (IRL Press,Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes,(Academic Press, New York, 1992); Guthrie and Fink, Guide to YeastGenetics and Molecular Biology (Academic Press, New York, 1991); Harlowand Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1998); Jakoby and Pastan, 1979; Nucleic Acid Hybridization(B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell AndMolecular Biology (Mayer and Walker, eds., Academic Press, London,1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andC. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition,(Blackwell Scientific Publications, Oxford, 1988); Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), 4th Ed., (Univ.of Oregon Press, Eugene, Oreg., 2000).

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of, orconsist of its particular stated element, step, ingredient, orcomponent. Thus, the terms “include” or “including” should beinterpreted to recite: “comprise, consist of, or consist essentiallyof.” As used herein, the transition term “comprise” or “comprises” meansincludes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient, or component not specified. The transitional phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients, or components and to those thatdo not materially affect the embodiment.

Unless otherwise indicated, all numbers used in the specification andclaims are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. When furtherclarity is required, the term “about” has the meaning reasonablyascribed to it by a person skilled in the art when used in conjunctionwith a stated numerical value or range, i.e. denoting somewhat more orsomewhat less than the stated value or range, to within a range of ±20%of the stated value; ±19% of the stated value; ±18% of the stated value;±17% of the stated value; ±16% of the stated value; ±15% of the statedvalue; ±14% of the stated value; ±13% of the stated value; ±12% of thestated value; ±11% of the stated value; ±10% of the stated value; ±9% ofthe stated value; ±8% of the stated value; ±7% of the stated value; ±6%of the stated value; ±5% of the stated value; ±4% of the stated value;±3% of the stated value; ±2% of the stated value; or ±1% of the statedvalue.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or illustrative language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to publications,patents, and/or patent applications (collectively “references”)throughout this specification. Each of the cited references isindividually incorporated herein by reference for their particular citedteachings.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the examples or when application of themeaning renders any construction meaningless or essentially meaningless.In cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

What is claimed is:
 1. A conotoxin peptide comprising the formula of SEQID NO:10.
 2. A conotoxin peptide of claim 1, comprising the formula ofSEQ ID NO:11.
 3. A conotoxin peptide of claim 2, comprising the formulaof SEQ ID NO:12.
 4. A conotoxin peptide comprising the formula of anyone from: SEQ ID NO:13-20.
 5. A conotoxin peptide comprising the formulaof any one from: SEQ ID NO:174-185.
 6. A conotoxin peptide of any ofclaims 1-3, wherein the C-terminus of the peptide is an amide group(—NH2).
 7. A conotoxin peptide of any of claims 1-3, wherein the peptideis linked to a fatty acid.
 8. A conotoxin peptide of claim 7, whereinthe fatty acid is a 3 to 60 carbon fatty acid.
 9. A conotoxin peptide ofany of claims 1-3, wherein the amino acid at the C terminus of theconotoxin peptide is replaced by the D-amino acid stereoisomer.
 10. Aconotoxin peptide of any of claims 1-3, wherein the N-terminal aminoacid is an acetylated amino acid.
 11. A conotoxin peptide of any ofclaims 1-3, wherein the peptide is biotinylated.
 12. A conotoxin peptideof any of claims 1-3, wherein the peptide is methylated.
 13. A conotoxinpeptide of any of claims 1-3, wherein the peptide is phosphorylated atone or more sites.
 14. A conotoxin peptide of any of claims 1-3, whereinthe peptide is glycosylated.
 15. A conotoxin peptide of any of claims1-3, wherein the peptide is linked to a fluorescent dye or a fluorescentprotein.
 16. A conotoxin peptide of any of claims 1-3, wherein twocysteine residues are each replaced with a natural or unnatural aminoacid that are then coupled for bridge formation.
 17. A conotoxin peptideof claim 16, wherein each of the cysteine residues is replaced with an(R)- or (S)-version of a naturally occurring amino acid selected fromaspartic acid, glutamic acid or lysine.
 18. A conotoxin peptide of claim16, wherein a first of the two cysteine residues is replaced with anunnatural amino acid containing carboxylic acid in a side chain and asecond of the two cysteine residues is replaced with an unnatural aminoacid containing an amine group in a side chain.
 19. A conotoxin peptideof claim 16, wherein each of the cysteine residues is replaced with(S)-propargyl glycine or (S)-azidonorvaline.
 20. A conotoxin peptide ofclaim 16, wherein the bridge is a lactam bridge or a triazole bridge.21. A conotoxin peptide of any of claims 1-3, wherein a linker isintroduced so as to generate an N-terminus to C-terminus cyclizedpeptide.
 22. A conotoxin peptide of claim 21, wherein the linkerconsists of a sequence of 1 to 100 amino acids.
 23. A conotoxin peptideof claim 21, wherein the linker is non-peptidic.
 24. A conotoxin peptideof any of claims 1-3, wherein the peptide is linked to polyethyleneglycol polymers.
 25. A conotoxin peptide of any of claims 1-3, whereinthe peptide is expressed as a fusion to a protein.
 26. A conotoxinpeptide of claim 25, wherein the protein is the Fc portion ofimmunoglobulin G (IgG).
 27. A pharmaceutical composition comprising theconotoxin peptide of any of claims 1-3.
 28. A pharmaceuticallyacceptable salt comprising the conotoxin peptide of any of claims 1-3.29. A method for treating at least one condition associated with theα9α10 subtype of the nicotinic acetylcholine receptor (nAChR) in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a conotoxin peptide, a compositioncomprising the conotoxin peptide, or a pharmaceutically acceptable saltcomprising the conotoxin peptide, wherein the conotoxin peptide is theconotoxin peptide of any of claims 1-3, thereby treating the condition.30. A method of claim 29, wherein at least one condition is pain.
 31. Amethod of claim 30, wherein the pain is general pain, chronic pain,neuropathic pain, nociceptive pain, inflammatory pain, pain induced byperipheral nerve damage, pain induced by an inflammatory disorder, paininduced by a metabolic disorder, pain induced by cancer, pain induced bychemotherapy, pain induced by a surgical procedure, and/or pain inducedby a burn.
 32. A method of claim 31, wherein the pain is cancer-relatedchronic pain and/or cancer-related neuropathy.
 33. A method of claim 29,wherein the at least one condition is an inflammatory condition.
 34. Amethod of claim 33, wherein the inflammatory condition is inflammation,chronic inflammation, a rheumatic disease, sepsis, fibromyalgia,inflammatory bowel disease, sarcoidosis, endometriosis, uterinefibroids, an inflammatory skin disease, an inflammatory condition of thelungs, a disease associated with inflammation of the nervous system,periodontal disease, or cardiovascular disease.
 35. A method of claim34, wherein the inflammatory condition is mediated by immune cells. 36.A method of claim 35, wherein the inflammatory condition is long-terminflammation and peripheral neuropathy following injury.
 37. A method ofclaim 29, wherein the at least one condition is pain and inflammation.38. A method of claim 29, wherein the at least one condition isinflammation and neuropathy.